An “adjuvant” is a substance that itself is often biologically inactive but which, in conjunction with a given antigen, enhances its antigenic capabilities. An adjuvant plays key role in promoting immune responses by accelerating or amplifying one or more specific phases of various immune responses. One advantage of the use of adjuvants in vaccines lies in the fact that the same degree of antibody response can be achieved with a smaller amount of antigen. Adjuvants are particularly useful in cases where the antigen alone does not stimulate high levels of antibody. Thus, the main functions of an adjuvant is to enhance the antibody response to levels which will ensure protection against an infectious disease. An ideal adjuvant should also up regulate the expression of cytokines responsible for T-helper cell activation essential for evoking immune memory and cell mediated immunity. Any material used as an adjuvant in vaccines should be non-toxic, relatively easily metabolized and produce little or no skin reaction at the injection site. Adjuvants have been used experimentally in animals for studies on the immune system and in vaccines for improvement of immunological responses and the enhancing effects have, in many instances, been confirmed by application to humans. Prior art adjuvants range in type from simple inorganic materials, such as, aluminum phosphate, to complex mixtures, such as, Freund's adjuvant, which is a homogenate of oil, detergent and killed tubercle bacilli.a\Typical examples of such adjuvants are aluminum hydroxide or phosphate or calcium salts (alum), non-ionic block polymer surfactants, an oil emulsion (Freund's adjuvant), and emulsion encompassing pathogen associated molecular patterns such as monophosphoryl lipid A (MPL), lipopolysaccharides, mycobacteria, muramyl dipeptides (MDP) tetanus toxoid, CpG, etc. plant derived Quillaja saponins (QS21).
Since the use of a protein antigen alone does not necessarily induce an adequately strong immune response, vaccine compositions normally contain an antigen in combination with an adjuvant. Adjuvants provide additional signal required by the antigen generated by the interaction with co-stimulatory molecule(s). In this regard, the adjuvant may be able to strengthen the signal generated by co-stimulatory molecules such as CD 40. CD 80, and CD 86 on antigen presenting cell (DC). It also induces MHC molecules and specific cytokines leading to Th1 activation and help in achieving the required level of TH1/Th2 bias for any specific vaccine. Th1 type immune response leads to the increase of IgG2a generation and induces a powerful cell mediated immune response.
Some antigens such as lipoproteins, glycoproteins, or whole microorganisms can act both as an epitope and an adjuvant in the form of a pathogen associated molecular pattern (PAMP). Even though the primary structure of a protein antigen, cannot be altered, the PAMP moiety of an antigen can be modified through the addition of a specific adjuvant or related immune potentiators to influence immunogenicity (Dempsey P W et al., Science 271: 348-350, 1996; Deres K et al, Nature 342: 561-564, 1989). Thus, the modification of a molecular pattern of an antigen can increase immunogenicity and help to achieve qualitatively specific immune response. (Milich D R et al., 1986, New Approaches to Immunization, pp 377-382. Cold Spring Harbor Laboratories, New York). The stimulation of Toll-Like Receptors (TLRs)present at the surface of antigen presenting cells (APCs) is now an established approach to triggering and boosting the immune response. Many compounds have been described as agonists of the TLRs, especially the TLR2 subtype [2], and as a consequence, can be considered as potential immune adjuvants. For example, very complex lipopeptides like macrophage-activating lipopeptide-2 [3](MALP-2)analogs i.e. Pam2Cys-GDPKHPKSF [(a) Akira, S et al. Cell 2006, 124, 783-801.]. Pam2Cys-GDPKHPKSFTGWVA representing the N-terminal part of the 44-kDa lipoprotein LP44 of Mycoplasma salivarium [Basith, S et al. Expert Opin. Ther. Patents 2011, 21, 927-944.], or Braun lipoprotein [x] have all been described as TLR2 agonists. On the other hand, simpler synthetic S-[2,3-bispalmitoyloxy-(2R)-propyl]-Rcysteinyl lipopeptides, like ‘Pam2CAG’ or Pam3CSK4 [Berg, M et al. American Journal of Physiology 1994, 266, 1684-1691], have also shown TLR2 agonist activity and can therefore be considered as more useful and easily attainable immunoadjuvants. In these constructs, the Pam2C (or Pam3C) template is necessary but not sufficient to stimulate the TLR2 receptor and a substitution of the cysteine is required to establish agonist activity. While lysine derivatives generally improve the activity of TLR2/6 agonists, it was also shown that Pam2CSK, the smallest lysine-containing compound, has no cytokine-inducing activity [Bessler, W. G et al Journal of Immunology 1985, 135, 1900-1905]. In this context, two lysines are usually needed to recover activity, which confer positive charges and above all highly amphiphilic properties that complicate the preparation of the vaccine cocktail. Recently, Pam2CS and its analogs have been synthesized and have shown good NF-kB inducing activity [Sinéad M. Smith et al J. Immunology, 2011, 186, 4, 2462-2471]. Nevertheless, their effects on DC and B-lymphocyte maturation and cytokine secretion have not yet been demonstrated. Glycolipids of the type Pam2Cys-α-Galactose as immunoadjuvants has been reported to have several folds increase in cell proliferation and DC maturation attributes when comparable with standard Pam2cysCAG or Pam3cysCAG.
A lipopeptide was first synthesized by Metzger et al. as a synthetic analogue of lipopeptide originated from bacteria and mycoplasma (Metzger J et al., Int J Peptide Protein Res 37:46-57, 1991). Since then, numerous analogues have been synthesized (EMC microcollections GmbH Sindelfinger Str. 3 72070 Tubingen. Germany). There is a report that virus-specific cytotoxic T lymphocyte (CTL) was induced by administrating a mouse with Pam3Cys-Ser-Ser, a lipopeptide conjugated with influenza virus T cell epitope (Schild H et al., Eur J Immunol 21:2649-2654, 1991). In general, the lipopeptide has been known as a TLR 2 ligand (Trinchieri G & Sher A, Nat Rev Immunol 7:179-190, 2007). These types of natural adjuvants associated with the antigens, however, often may not be strong enough to induce a desired strength and a quality of immune response, requiring a good adjuvant in a vaccine formulation.
Developing a good adjuvant is accordingly a very important job in developing a good vaccine, but adjuvant development still has to rely mainly on empirical work. For example, Toll Like Receptors (TLR)are the most important PRR on antigen presenting cells (APC) involved in the activation of APC and in antigen presentation by APC. Potent antibody response, however, is not entirely dependent on TLR signals (Gavin. A. L. et al, Science 314:1936-1938, 2006). Further. Pam3cys, which is a TLR2 ligand, works in inducing immune response independently of TLR2 (Yoder et al. Infect. Immun. 71:3894-3900, 2003). Accordingly, although Pam2Cys and a galactosyl moiety covalently linked together is known to be synergistic in inducing antibody and cytokine response (Bagchi et at J. Immun. 178:1164-1171, 2007), there has been no prior art of a well balanced powerful vaccine adjuvant function of a bioconjugate encompassing any carbohydrate moiety and a di or triacyl cystenyl lipid head group linked through a 1,2,3-triazolyl methyl amine spacer. Since, good protective immunity requires balanced immune response comprising both strong cell mediated immune response and humoral antibody response, developing an adjuvant that aid such powerful and synergestic immune response is an important endeavor in vaccine research.